Summary of work: Genotoxic/oxidative stresses contribute to the development of degenerative diseases, and may underlie the aging process itself. Cells respond to such stresses with the induction of numerous gene products, but much remains to be learned concerning the signaling pathways mediating these effects or the functional significance of the induced gene products. This project encompasses studies related to these cellular responses. Efforts over the past year have been focused in three main areas: 1. Activation of mitogen-activated protein kinases (MAPK) in response to genotoxic/oxidative stress. Using chemical inhibitors selective for specific MAPKs, or dominant negative mutants of specific mediators of the pathways, we have examined the importance of ERK, JNK and p38 MAPK, for survival following oxidant injury. In general, we have found that elevated ERK activity is associated with survival, while elevated JNK is correlated with enhanced cytotoxicity. Although p38 is highly induced by oxidant injury, it does not influence survival. We have also investigated the response to oxidative/genotoxic stress as a function of aging using primary hepatocytes derived from young and aged animals. We have provided evidence that ERK activation in response to several stresses including hydrogen peroxide, arsenite and heat is attenuated in cells of aged animals and this correlated with reduced survival. 2. Role of posttranscriptional events in gene induction of stress response genes. We have found that induction of the cyclin dependent kinase inhibitor p21Waf1/Cip1 mRNA occurs through changes in mRNA stability. This is dependent on the presence of p53 tumor suppressor protein and involves a tyrosine kinase/phosphatase regulatory system. Future studies will address the nature of the p53 dependency, and the specific kinases/phosphatases involved. 3. Up-regulation of the cholesterol biosynthetic pathway during the cellular response to stress. We have provided evidence that UVC irradiation and heat stress lead to elevated synthesis of mevalonate and mevalonate-derived sterols. This constitutes a previously unrecognized stress-response pathway that could intersect with MAPK cascades, as critical mediators of the ERK and JNK pathways (e.g. small GTP-binding proteins such as Ras and Rac) are dependent on mevalonate-derived isoprenoids for their membrane association and activation.